Inflammatory reactions, autoimmune and autoinflammatory diseases, and cardiovascular diseases such as atherosclerosis or myocardial ischemia characteristically feature local inflammatory processes which initially aim for repair of injured tissue, but in effect cause damage to tissue during chronic diseases. Cells of the innate immune system, especially pro-inflammatory activated phagocytes such as macrophages or neutrophils, but also epithelial cells, play a pivotal role in many inflammatory disorders. Activated phagocytes significantly contribute to the progression of inflammatory diseases such as atherosclerosis, myocardial infarction, acute coronary syndromes, autoimmune diseases as rheumatoid arthritis, inflammatory bowel diseases, infectious diseases, tumors and others.
In various diseases at sites of inflammation, e.g. in inflammatory atherosclerotic plaques, activated phagocytes and epithelial cells express and locally secrete high levels of the S100 protein complex S100A8/S100A9, also known as calprotectin, which acts as so called alarmin or Danger Associated Molecular Pattern (DAMP) molecule with potent pro-inflammatory capacities (Vogl et al. 2007, Loser et al. 2010, Chan et al. 2012). These bind to both the extracellular matrix and receptors such as Toll-like receptor 4 (TLR4) or receptor for advanced glycation endproducts (RAGE) on the immune cell surface, thereby amplifying inflammatory reactions. A correlation between serum levels of S100A8/S100A9 and disease activity has been observed in many inflammatory diseases (Foell and Roth, 2004). Moreover, overexpressed S100A8/S100A9 reflects the disease activity in many inflammatory cardiovascular disorders. For example, in acute coronary syndrome (ACS) or atherosclerotic plaques in instable angina pectoris, S100A9 belongs to the highest of upregulated genes. Here S100A8 and S100A9 constitute 40% of neutrophilic and up to 5% of monocytic cytosolic protein (Hessian et al. 1993). S100A9−/− mice are protected from endotoxin-induced lethal shock and E. Coli-induced abdominal sepsis, indicating strong pro-inflammatory functions of these proteins. The expression of S100A9 and S100A8 is generally regarded as closely related, and the loss of S100A9 results in a functional knockout of S100A8 protein in S100A9 deficient mice.
Since both S100A8 and S100A9 proteins are actively secreted by activated cells at site of inflammation and participate in pro-inflammatory cascades, they are considered as attractive targets for non-invasive molecular imaging, especially since despite tremendous efforts in blood testing and conventional clinical imaging the monitoring of the local activity of these inflammatory processes remains unsatisfactory. Conventional imaging techniques are useful to show structural damage due to chronic inflammation, but still lack the necessary cellular and molecular specificity and sensitivity to detect inflammatory disease activity itself. In contrast, molecular imaging techniques such as single photon emission tomography (SPECT) and positron emission tomography (PET), relying on the intravenous application of radiopharmaceuticals, are capable of offering unique molecular sensitivity for preclinical and clinical studies. Thus, these technologies allow for the in vivo diagnosis of inflammatory diseases on the basis of anatomical, morphological and physiological changes and to assess efficiency of therapy.
Previously, the use of antibody-based optical probes such as Cy5.5®-labelled antibodies targeting S100A9 (anti-S100A9-Cy5.5) has been reported in disease scores in vivo and allowed for excellent correlations of signal intensity and successful imaging of inflammatory activity in various mouse models of inflammation, e.g. allergic and toxic contact dermatitis, collagen induced arthritis, and Leishmania major infection (Vogl et al. 2014). Using fluorescence reflectance imaging (FRI), Cy5.5®-labelled antibodies could be effectively applied for non-invasive imaging and the detection of S100A9-expression at the local site of inflammation. However, translation of this imaging strategy into patients is a challenge due to known limitations of antibody imaging.
Moreover, a novel class of non-peptidic compounds (quinoline-3-carboxamide compounds, which are also denoted as Q-compounds) having strong binding affinity against S100A9 has been identified for the treatment of autoimmune diseases (Bjork et al. 2009). Applying photoaffinity labelling, FITC-labelled quinoline-3-carboxamide compounds have been used as probes for “fishing” cells expressing S100A9 as target on their surface. The binding affinity of FITC-labelled quinoline-3-carboxamide compounds to human peripheral blood mononuclear cells (PBMC) could be demonstrated, and S100A9 as most prominent binding protein has been identified in the cell membrane applying matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF). Using recombinant S100A9 and S100A8, a strong binding affinity of quinoline-3-carboxamide compounds to homodimeric S100A9 could be observed, whereas only weak binding was observed for the S100A8/S100A9 complex, and close to baseline levels for S100A8 homodimer. Thus, Björk et al. 2009 conclude that binding of quinoline-3-carboxamide compounds is more or less exclusively restricted to homodimeric S100A9 and thus, quinoline-3-carboxamide compounds have high potency in inhibiting the interaction between human and mouse S100A9 and TLR4/MD2. Accordingly, S100A9 may be a potential pharmacological target for quinoline-3-carboxamide compounds, and the use of quinoline-3-carboxamide compounds in the treatment of autoimmune/inflammatory diseases has been suggested.
Although labelled S100A9 antibodies for use in a method of diagnosing inflammatory diseases have been described in the art in mice, there exists a need for alternative compounds having high molecular specificity to S100A9 for a simple in vivo diagnosis of inflammatory diseases associated with an increased release of S100A9 in humans for use in in preclinical and clinical trials. Such compounds should be suitable to analyze molecular levels of already early stage inflammation at local site, and should be applicable for other non-invasive imaging modalities as for example single photon emission tomography (SPECT), positron emission tomography (PET), optical imaging, ultrasound and photoacoustic imaging, which offer unique molecular sensitivity. Diagnosing inflammatory diseases at already early states would be a great advantage for the prognosis of patients and appropriate treatments could be started in time. Additionally, such compounds should be suitable to evaluate the risk of a subject to develop an inflammatory disease and to monitor the progression of an inflammatory disease under different therapeutic conditions. Accordingly, there is a need in the art for compounds having high molecular sensitivity to S100A9 for the prognosis of inflammatory diseases and for monitoring the effectiveness of inflammatory disease treatment.
In sum, there is a need in the state of the art to provide new means and methods that help to diagnose inflammatory disease associated with an increased accumulation of S100A9 at local site of inflammation at molecular level. The technical problem underlying the present application is thus to comply with this need. The technical problem is solved by providing the embodiments reflected in the claims, described in the description and illustrated in the examples and figures that follow.